1. Field of the Invention
The present invention relates generally to genotyping CCR alleles in individuals and populations. More specifically, the present invention relates to methods of correlating CCR alleles and/or genotypes, specifically CCR5 promoter alleles, with HIV-1 transmission and/or disease progression.
2. Description of the Related Art
xe2x80x9cChemokinexe2x80x9d describes a closely related family of xe2x80x98chemotactic cytokinesxe2x80x99 with conserved sequences, known to be potent attractors for various leukocyte subsets such as neutrophils, monocytes, or lymphocytes. Chemokines are a large superfamily consisting of four subfamilies that display between two and four highly conserved NH2-terminal cysteine amino acid residues. The CXC (or xcex1) family has the first two NH2-terminal cysteines separated by one nonconserved amino acid residue. In contrast, the CC (or xcex2) family has these cysteines in juxtaposition, and the C (or xcex3) family has one lone NH2-terminal cysteine residue, while the CX3C (or xcex4) family has these cysteines separated by three intervening amino acids. The large number of chemokines and their receptors, together with the expression of chemokine receptors on cells other than leukocytes (such as epithelial, endothelial and smooth muscle cells) is indicative of the importance of these molecules.
Six receptors for the CC family of chemokines have been identified: CC-CCR1, CC-CCR2, CC-CCR3, CC-CCR4, CC-CCR5 and the duffy blood group antigen. These proteins are seven transmembrane domain G protein-coupled receptors. In general, there is broad overlap in the ligands bound by the CC chemokine receptors. The human CCR5 and CCR2 chemokine receptor genes, which serve as co-receptors with CD4 for HIV, are tightly linked on chromosome 3p21-22, separated by 20 kb.
The complex mechanisms for HIV-1 entry into human CD4+ cells reflect in part its evolving usage of co-receptors (35). For example, macrophage-tropic HIV-1 isolates infect cells by binding to the CD4+ receptor and the CCR5 co-receptor (36), while T-cell-tropic viruses mainly use CXCR4 as the co-receptor (37). HIV-1 represents a close relative of simian immunodeficiency virus (SIV) that is naturally found in African primates, including chimpanzees and sooty mangabeys (15). Recent studies have confirmed that HIV-1 originated in the chimpanzee subspecies Pan troglodytes troglodytes from Central West Africa (16). Both HIV and SIV prefer CCR5 as their co-receptor for penetrating CD4+ cells (17-19), which suggests that CCR5 is commonly expressed in both human and non-human primates. Therefore, highly conserved transcription factor binding elements may represent a critical mechanism for regulating transcription of the CCR5 gene, and may serve as appropriate targets for intervention experiments. Additionally, other members of the CCR co-receptor gene family also facilitate viral transmission (38), but they appear to have more restricted cellular distribution (19,39-40).
Recent work in AIDS cohorts has revealed associations between several polymorphisms in the CC (beta) chemokine receptor loci and either variable degrees of protection against HIV-1 transmission or variable evolution of the AIDS that follows (1-4). In Caucasians, homozygosity for a 32-bp deletion (xcex9432) mutation in the CCR5 coding sequence leads to the absence of cell surface expression of CCR5, which confers nearly complete resistance to HIV-1 infection (1,41-44). Another less frequent mutation at nucleotide position 303 of the CCR5 coding sequence (m303) introduces a premature stop codon that also abolishes surface expression of CCRS in Caucasians. Accordingly, an individual carrying both CCR5-xcex9432 and m303 is resistant to HIV-1 infection (45). Carriage of a single copy of CCR5-xcex9432 in the presence of CCR5 wild-type provides little if any resistance to HIV-1 infection, but does tend to delay the progression of disease (1,4,46), possibly through interference with translocation of the wild-type product (47). Neither the CCRS-xcex9432 nor the CCR5-m303 allele is frequent in African and Asian populations (1,43,48). However, African and Asian ethnic groups also show great variability in acquisition and progression of the disease. Population-specific polymorphisms within the CCRS gene may exist (49), but none of these have been associated with varying degrees of viral susceptibility (48). The use of CCRS as the co-receptor is not altered whether or not additional CCRS polymorphisms are present, regardless of the HIV-1 subtype (50).
In addition, extensive heterogeneities have been described in the expression and splicing of CCR5, apparently regulated by different CCR5 promoter alleles (3). More importantly, 2 CCR5 promoter variants have been associated with contrasting rates of HIV-1 disease progression (5,6). Expression of cell surface CCR5 also varies widely, even in individuals homozygous for wild type CCR5 (51,52), indicating differential regulation of CCR5 production. Therefore, variability in infectivity of primary HIV-1 isolates that prefer CCR5 as the co-receptor is observed even in wild type individuals. Detailed study of the organization of the CCR5 gene and its promoter has uncovered extensive heterogeneity in the 5xe2x80x2 untranslated region (UTR) of CCR5 mRNA (13). Some of these polymorphisms may be linked to changes in splicing and may account for continuing uncertainties about the intron and exon boundaries (13-14,53). One CCR5 promoter variant (59653T) shows strong linkage disequilibrium with CCR2b-64I (2)xe2x80x94a CCR variant previously associated with retarded disease progression (4), and subsequent work has further detailed the relationships of CCR5 promoter markers to both CCR2b and CCR5-xcex9432 (3,5,6). More recent association of the 59029G/G genotype with reduced promoter activity (5) also suggests that CCR5 promoter polymorphisms independently modulate HIV-1 disease progression.
The mechanisms and credibility of these specific genetic associations are under debate (3,10), especially because some of the CCR variants are tightly linked to each other (2). However, these findings highlight the importance of host genetic factors in HIV/AIDS and may guide development of new measures for the prevention and control of HIV-1-related diseases (7-9).
Polymorphisms at the CCR2b, CCR5, and CCR5 promoter loci have been analyzed in four ethnic groups, with a special emphasis on the relationships of CCR5 promoter allelic variants to other well-characterized markers previously associated with different outcomes of HIV-1 transmission and disease progression. In addition, the same CCR5 promoter polymorphisms were analyzed in HIV-1-infected Rwandan women, with a special emphasis on the influence of the genetic polymorphisms on HIV-1 disease progression.
The prior art is deficient in methods used to predict the likelihood and/or probability of HIV-1 transmission and/or disease progression based upon CCR alleles and/or genotypes. The present invention fulfills a long-standing need for the development of a rapid and informative genotyping strategy that can be readily applied to analyze CCR2, CCR5 and related genetic variants and to evalulate the relationship of each genotype to HIV transmission and disease progression.
Variability in HIV-1 infection has been associated with genetic variants in the beta-chemokine receptor 5 (CCR5) locus. Genetic variations (a 32-bp deletion and a point mutation) in the coding sequence of the HIV-1 co-receptor, CCR5, have been shown to confer resistance to HIV-1 infection by depleting CCR5 expression on the cell surface. While CCR5 coding sequences have exhibited relatively limited variation, its promoter sequence appears polymorphic in all major populations.
The studies reported herein revealed five major CCR5 promoter alleles with distributions that differed widely among the four distinct ethnic groups examined. Herein, the methods of the instant invention are used to present evidence that particular genetic variants of the CCR5 promoter appear to determine the infectability of an individual (i.e., herein, of heterosexual women in Kigali, Rwanda, and injecting drug users in New York, USA). Additionally, the present methods have been used to examine the relationship between the major CCR5 promoter genotypes and HIV-1 to AIDS disease progression (i.e., among 201 HIV-1-infected Rwandan women). The effects of disease progression-related CCR2b and CCR5 polymorphisms on early HIV-1 viral load were also determined in a cohort of homosexual HIV-1 seroconverters. The methods of the present invention allowed the inventors to establish an independent and strong linkage between HIV-1 transmission and CCR5 promoter alleles using these distinct cohorts and further, to validate the methodology of the present invention.
One object of the present invention is to provide methods of correlating CCR alleles and/or genotypes, specifically CCR5 promoter alleles, with HIV-1 transmission and/or disease progression.
In an embodiment of the present invention, there is provided a method of surveying CCR genotypes in a population, comprising the steps of: (a) obtaining biological samples from a representative number of individuals in a population, with each sample being from a different individual, wherein the sample contains genomic DNA; (b) combining a portion of each sample with at least one experimental primer combination and a control primer combination, thereby producing primer-annealed DNA, wherein the experimental primer combinations are selected from the group consisting of SEQ ID Nos. 2 and 3, 2 and 4, 5 and 7, 5 and 8, 5 and 9, 5 and 10, 5 and 11, 5 and 12, 5 and 13, 6 and 7, 6 and 8, 6 and 12, 6 and 13, 14 and 16 and 15 and 16, wherein the control primer combination is SEQ ID Nos. 17 and 18; (c) amplifying the primer-annealed DNA in a reaction, thereby producing amplicons, wherein reaction conditions for the amplification are optimized for sequence-specific amplification, wherein each experimental primer combination and the control primer combination are predicted to produce one or more amplicons having expected sizes in basepairs; (d) separating the amplicons by size, wherein the presence of: a 197 bp amplicon with the control primer combination is indicative of a CCR5 wildtype coding sequence; a 165 bp amplicon with the control primer combination is indicative of a CCR5-xcex9432 coding sequence; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 7, a 367 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 10, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 12 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a P*0101 CCR5 promoter allele; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 7, a 367 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 10, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 13 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a P*0102 CCR5 promoter allele; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 7, a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 9, a 367 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 11, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 13 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a P*0103 CCR5 promoter allele; a 363 b p amplicon with experimental primer combination SEQ ID Nos. 6 and 8, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 13 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a P*0201 CCR5 promoter allele; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 8, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 13 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 15 and 16 is indicative of a P*0202 CCR5 promoter allele; (e) determining a CCR genotype for each sample based upon the CCR alleles indicated following step (d); and (f) compiling the genotypes determined in step (e), thereby genotyping the representative number of individuals in the population, thereby surveying CCR genotypes in the population.
In another embodiment of the present invention, there is provided a method of surveying HIV-1 co-receptor CCR alleles in an individual, comprising the steps of: (a) obtaining a biological sample from an individual, wherein the sample comprises genomic DNA; (b) combining a portion of the sample with at least one experimental primer combination and a control primer combination, thereby producing primer-annealed DNA, wherein the experimental primer combinations are selected from the group consisting of SEQ ID Nos. 2 and 3, 2 and 4, 5 and 7, 5 and 8, 5 and 9, 5 and 10, 5 and 11, 5 and 12, 5 and 13, 6 and 7, 6 and 8, 6 and 12, 6 and 13, 14 and 16 and 15 and 16, wherein the control primer combination is SEQ ID Nos. 17 and 18; (c) amplifying the primer-annealed DNA in a reaction, thereby producing amplicons, wherein reaction conditions for the amplification are optimized for sequence-specific amplification, wherein each experimental primer combination and the control primer combination are predicted to produce one or more amplicons having expected sizes in basepairs; and (d) separating the amplicons by size, wherein the presence of: a 197 bp amplicon with the control primer combination is indicative of a CCR5 wildtype coding sequence; a 165 bp amplicon with the control primer combination is indicative of a CCR5-xcex9432 coding sequence; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 7, a 367 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 10, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 12 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a P*0101 CCR5 promoter allele; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 7, a 367 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 10, a 412 b p amplicon with experimental primer combination SEQ ID Nos. 5 and 13 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a P*0102 CCR5 promoter allele; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 7, a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 9, a 367 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 11, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 13 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a P*0103 CCR5 promoter allele; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 8, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 13 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a P*0201 CCR5 promoter allele; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 8, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 13 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 15 and 16 is indicative of a P*0202 CCR5 promoter allele.
In yet another embodiment of the present invention, there is provided a method of predicting the disease progression to AIDS in an HIV-1-infected individual, comprising the steps of: (a) obtaining a biological sample from an individual, wherein the sample comprises genomic DNA; (b) combining a portion of the sample with at least one experimental primer combination and a control primer combination, thereby producing primer-annealed DNA, wherein the experimental primer combinations are selected from the group consisting of SEQ ID Nos. 2 and 3, 2 and 4, 5 and 7, 5 and 8, 5 and 9, 5 and 10, 5 and 11, 5 and 12, 5 and 13, 6 and 7, 6 and 8, 6 and 12, 6 and 13, 14 and 16 and 15 and 16, wherein the control primer combination is SEQ ID Nos. 17 and 18; (c) amplifying the primer-annealed DNA in a reaction, thereby producing amplicons, wherein reaction conditions for the amplification are optimized for sequence-specific amplification, wherein each experimental primer combination and the control primer combination are predicted to produce one or more amplicons having expected sizes in basepairs; (d) separating the amplicons by size, wherein the presence of: a 363 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 8, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 13 and a 309 b p amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a CCR5 promoter genotype of P*0201/P*0201; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 7, a 367 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 10, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 13, a 363 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 8, a 412 b p amplicon with experimental primer combination SEQ ID Nos. 6 and 13, a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 15 and 16 is indicative of a CCR5 promoter genotype of P*0102/P*0202; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 7, a 367 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 10, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 12, a 363 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 8, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 13 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a CCR5 promoter genotype of P*0101/P*0201; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 7, a 367 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 10, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 12, a 363 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 8, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 13, a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 15 and 16 is indicative of a CCR5 promoter genotype of P*0101/P*0202; wherein a CCR5 promoter genotype of P*0201/P*0201 or P*0102/P*0202 is predictive of an accelerated rate of AIDS progression in the individual (relative to an individual who does not possess the P*0201/P*0201 or P*0102/P*0202 gentoype), wherein a CCR5 promoter genotype of P*0101/P*0201 or P*0101/P*0202 is predictive of a slower rate of AIDS progression in the individual (relative to an individual who does not possess the P*0101/P*0201 or P*0101/P*0202 gentoype).
In still yet another embodiment of the present invention, there is provided a method of predicting the probability of HIV-1 infection in an individual, comprising the steps of: (a) obtaining a biological sample from an individual, wherein the sample comprises genomic DNA; (b) combining a portion of the sample with at least one experimental primer combination and a control primer combination, thereby producing primer-annealed DNA, wherein the experimental primer combinations are selected from the group consisting of SEQ ID Nos. 2 and 3, 2 and 4, 5 and 7, 5 and 8, 5 and 9, 5 and 10, 5 and 11, 5 and 12, 5 and 13, 6 and 7, 6 and 8, 6 and 12, 6 and 13, 14 and 16 and 15 and 16, wherein the control primer combination is SEQ ID Nos. 17 and 18; (c) amplifying the primer-annealed DNA in a reaction, thereby producing amplicons, wherein reaction conditions for the amplification are optimized for sequence-specific amplification, wherein each experimental primer combination and the control primer combination are predicted to produce one or more amplicons having expected sizes in basepairs; and (d) separating the amplicons by size, wherein the presence of: a 363 bp with experimental primer combination SEQ ID Nos. 6 and 8, a 412 bp with experimental primer combination SEQ ID Nos. 6 and 13 and a 309 bp with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a CCR5 promoter genotype of P*0201/P*0201; a 363 bp with experimental primer combination SEQ ID Nos. 5 and 7, a 367 bp with experimental primer combination SEQ ID Nos. 5 and 10, a 412 bp with experimental primer combination SEQ ID Nos. 5 and 12 and a 309 bp with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a CCR5 promoter genotype of P*0101/P*0101; wherein a CCR5 promoter genotype of P*0201/P*0201 is predictive of a decreased probability of HIV-1 infection in the individual (relative to an individual who does not possess the P*0201/P*0201 genotype), wherein a CCR5 promoter genotype of P*0101/P*0101 is predictive of an increased probability of HIV-1 infection in the individual (relative to an individual who does not possess the P*0101/P*0101 genotype).
In yet another embodiment of the present invention, there is provided a method of correlating CCR genotypes with HIV-1 transmission and/or disease progression, comprising the steps of: (a) obtaining biological samples from a representative number of individuals, wherein each sample is from a different individual, wherein the sample comprises genomic DNA; (b) assessing each individual""s HIV-1 status and/or risk of acquiring HIV-1; (c) assigning each individual to a risk group, wherein the assignment is based upon the individual""s HIV-1 status and/or risk of acquiring HIV-1; (d) combining a portion of each sample with at least one experimental primer combination and a control primer combination, thereby producing primer-annealed DNA, wherein the experimental primer combinations are selected from the group consisting of SEQ ID Nos. 2 and 3, 2 and 4, 5 and 7, 5 and 8, 5 and 9, 5 and 10, 5 and 11, 5 and 12, 5 and 13, 6 and 7, 6 and 8, 6 and 12, 6 and 13, 14 and 16 and 15 and 16, wherein the control primer combination is SEQ ID Nos. 17 and 18; (e) amplifying the primer-annealed DNA in a reaction, thereby producing amplicons, wherein reaction conditions for the amplification are optimized for sequence-specific amplification, wherein each experimental primer combination and the control primer combination are predicted to produce one or more amplicons having expected sizes in basepairs; and (f) separating the amplicons by size, wherein the presence of: a 197 bp amplicon with the control primer combination is indicative of a CCR5 wildtype coding sequence; a 165 bp amplicon with the control primer combination is indicative of a CCR5-xcex9432 coding sequence; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 7, a 367 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 10, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 12 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a P*0101 CCR5 promoter allele; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 7, a 367 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 10, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 13 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a P*0102 CCR5 promoter allele; a 363 b p amplicon with experimental primer combination SEQ ID Nos. 5 and 7, a 363 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 9, a 367 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 11, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 5 and 13 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a P*0103 CCR5 promoter allele; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 8, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 13 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 14 and 16 is indicative of a P*0201 CCR5 promoter allele; a 363 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 8, a 412 bp amplicon with experimental primer combination SEQ ID Nos. 6 and 13 and a 309 bp amplicon with experimental primer combination SEQ ID Nos. 15 and 16 is indicative of a P*0202 CCR5 promoter allele.
In a further embodiment of the present invention, there is provided an oligonucleotide selected from the group consisting of SEQ ID Nos. 2-16. Other aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.